Project Summary Abstract - R01ES016893 Hexavalent chromium (Cr(VI)), also known as chromate, is a major public health concern. Chromates, particularly the insoluble compounds, are well-established human lung carcinogens. Our study focuses on investigating the mechanisms of Cr(VI)-induced carcinogenesis, which are currently unknown. Recent studies indicate that particulate Cr(VI) induces chromosome instability, which is a hallmark of human lung tumors. However, how Cr(VI) induces chromosome instability is poorly understood. Our preliminary data show that chronic exposure to particulate Cr(VI) induces a DNA repair deficient phenotype that underlies the chromosome instability, and thus, the goal of this research is to characterize this repair deficiency and its impact and understand the mechanisms that cause it to occur. We will test the hypothesis that particulate Cr(VI) induces epigenetic changes inactivating homologous recombination repair of Cr(VI)-induced DNA double strand breaks resulting in increased chromosome instability and carcinogenesis. We will test this hypothesis through four interrelated specific aims. Aim 1 will characterize the homologous recombination repair defect and show that particulate Cr(VI) causes cells to inactivate Rad51-mediated response and switch to lower fidelity non-homologous end joining (NHEJ) repair using immunoblotting, immunofluorescence and repair assays. Aim 2 will show that the cells with particulate Cr(VI)-inactivated Rad51 response acquire chromosome instability and undergo neoplastic transformation using cytogenetic techniques and assays for contact- uninhibited and anchorage independent growth. Aim 3 will identify an epigenetic change (increased acetylation) that causes the loss of Rad51 response by affecting E2F1, ATM, Rad51C and Rad51 with assays for acetylation status, gene expression, and protein interactions and localization. Finally, Aim 4 will characterize the mechanism for particulate Cr(VI)-induced acetylation changes studying Cr(VI) binding to acetyl groups using assays for acetylation and histone deacetylase activity. Each aim will focus on human lung cells and confirm key findings in human Cr(VI) tumors. Results will lead to the first reports of detailed information of the interactions of Cr(VI with DNA double strand break machinery, acetylation status and the first characterizations of these aspects in tumors from Cr(VI)-exposed workers. This research is significant because it will provide: 1) An understanding of particulate Cr(VI)'s carcinogenic mechanism; 2) Essential information to better assess the risk of exposure to particulates; and 3) A mechanistic approach for further study of Cr(VI), other metals, and lung cancer in general.